Cardiovascular disease constitutes a major health burden globally, for which novel cardiotonic agents are still required. Cardiac failure is thought to be caused by dysfunctions of the sarcoplasmic/endoplasmic reticulum (SR/ ER) in cardiomyocytes. Therefore, in this study, we searched for novel pharmaceutical targets in SR/ER. Tissue and organelle specific proteome profiling by liquid chromatography coupled with mass spectrometry after gel electrophoresis separation identified 3,638 proteins in heart and liver SR/ER samples from a mouse transverse aortic constriction (TAC) model (heart failure). We also analyzed the transcriptome of heart tissue from the TAC model (heart failure, hypertrophy) and a myocardial infarction model using microarrays to identify differentially expressed genes in the diseased heart. Several genes were chosen for further studies following the proteome and transcriptome analyses. Of these, fat storage-inducing transmembrane proteins 1 and 2 (FITM1 and FITM2) were highly expressed in mouse and human heart and skeletal muscle. We investigated the functions of FITM1 and FITM2 in vitro and confirmed that they mediated lipid droplet (LD) formation and directly bound to triglycerides. FITM1 and/ or FITM2 overexpression in cells altered the levels of Ero1-Lα and PDI, which are ER stress marker proteins that protect against heart failure and affect cellular metabolism. Together, these results indicate that FITM1 and FITM2 are expressed in heart tissue and that their modulated expression or function can change LD formation, ER function, and cellular metabolism in cells. Thus, FITM1 and FITM2 are good drug target candidates.
Fat storage-inducing transmembrane proteins 1 and 2 (FITM1 and FITM2, respectively) are transmembrane endoplasmic/sarcoplasmic reticulum proteins involved in lipid droplet formation. The physiological functions of FITM1 have only been reported in skeletal muscle, while those of FITM2 were analyzed using genetically engineered mice. However, their roles in the heart have not been characterized. To examine their cardiac functions, we analyzed Fitm1- or Fitm2-knockout mice. Neither constitutive Fitm1 (−/−) aged nor heart failure model mice showed significant differences in heart size or function. Fitm2 (−/−) mice exhibited embryonic death, and aged Fitm2 (+/−) mice had shortened left ventricular end-diastolic dimension, and shortened left ventricular end-systolic dimension. However, body weight and ejection fraction of Fitm2 (+/−) mice were similar to those of wild-type littermates. In the chronic heart failure models, Fitm2 (+/−) mice showed significant suppression of increased left ventricular end-diastolic dimension and reduced ejection fraction. These results suggest the involvement of Fitm2 in chronic heart failure, whereas Fitm1 have a minor effect in this context in mice.
1594 Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory processes. As the major effectors, eosinophils function in a variety of biological responses, allergic diseases and helminth infections. It is generally accepted human eosinophils develop through a pathway initially sharing common feature with basophils. However, there lacks a clear chart for early development of human eosinophils, such as during embryonic or fetal stages. We recently established an efficient method for producing eosinophils from human embryonic and induced pluripotent stem cells (hESC/iPSCs). By a two-step induction, we first generated multipotential hematopoietic progenitors by co-culturing hESC/iPSCs with mouse AGM-derived stromal cells for 2 weeks. Then, total co-culture cells were transferred into suspension culture favoring eosinophil development with addition of IL-3 and other factors (SCF, IL-6, TPO, Flt-3 ligand) . The maturation of hESC/iPSC -derived eosinophils was shown in a time-dependent manner, first co-expressing eosinophil-and basophil-specific markers [eosinophil peroxidase (EPO), and 2D7, respectively], then the portion of eosinophil markers gradually increased while that of basophil markers decreased (EPO+ cells from 56.4% at day 7 to 94.4% at day 21, while 2D7+ cells from 62.8% to 25.7%, respectively), typically mimicking the development of eosinophils from human adult hematopoietic progenitors. By flowcytometric analysis, an eosinophil-specific surface marker, Siglec-8, was also expressed on these hESC/iPSC-derived eosinophils in a time-dependent manner (from 10.8% at day 7 to 91.3% at day 21), paralleling to those with EPO. The expression of eosinophil-specific granule cationic proteins (EPO, MBP, ECP, EDN) and IL-5 receptor mRNA was also detected by RT-PCR. Furthermore, transmission electron microscopy (TEM) observation confirmed the eosinophil property. Eosinophils derived from hiPSCs hold similar characteristics as those from hESCs. The function of hES/hiPSC-derived eosinophils is being under investigation. Our study provides an experimental model for exploring early genesis of eosinophils, especially in uncovering the mechanisms controlling the development of the initial innate immune system of human being in normal and diseased individuals. Disclosures: No relevant conflicts of interest to declare.
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